Filamentous fungi are a rich source of agriculturally and pharmaceutically important natural products. For example, the fungal polyketide statins, such as lovastatin, are among the most widely-prescribed drugs for the prevention and treatment of atherosclerosis by inhibiting cholesterol biosynthesis. Fungal polyketides belonging to the resorcylic acid lactone family exhibit potent antiproliferative activities as selective kinase inhibitors. Iterative fungal polyketide synthases (PKSs) use a unique set of biochemical rules in the synthesis of complex polyketides. These rules dictate polyketide starter unit selection, chain length control, and post-PKS processing. While the biosynthetic origins of bacterial polyketides have been studied extensively and have led to the combinatorial biosynthesis of pharmaceutically important unnatural natural products, the biosynthetic mechanisms of fungal PKSs are not well understood and their potential for combinatorial biosynthesis has not yet been realized. This is largely due to difficulties associated with manipulating these megasynthases in their native or related fungal hosts, and with obtaining intact enzymes for biochemical analysis. The objective of this proposal is to bridge these important knowledge and technical gaps and provide a multi- angled picture of the fungal polyketide biosynthesis employing the workhorse organism Escherichia coli. We have obtained extensive preliminary biochemical data on the expression, reconstitution and engineering of PKS4 from Gibberella fujikuroi (gfPKS4) and PKS13 from Gibberella zeae (gzPKS13) using E. coli as the heterologous host. This proposal will evaluate the following hypotheses: 1) Fungal PKS megasynthases can be functionally reconstituted in a bacterial host, such as E. coli;2) Fungal PKS contains initiation and cyclization domains that can be exploited for combinatorial biosynthesis;3) Fungal and bacterial catalytic components can be catalytically integrated towards the synthesis of novel polyketides. To address these hypotheses in a five-year period, we have defined the following three SPECIFIC AIMS: 1) Biochemical Characterization of gfPKS4 and gzPKS13 Initiation Domains;2) Biochemical Characterization of gfPKS4 and gzPKS13 Cyclization Domains and 3) Catalytic Integration of fungal and bacterial PKSs. Project Narrative We have proposed biochemical and metabolic engineering studies to investigate fungal polyketide synthases. Fungal polyketide synthases are iterative megasynthases that catalyze the biosynthesis of a number of biological active compounds, including those that are anticancer and antihypercholesterolemia. We will use the robust heterologous host Escherichia coli to study the initiation, elongation, termination and cyclization steps of the intact synthases. Knowledge gained from these studies will be valuable in the engineering of these enzymes towards synthesis of novel compounds both in vivo and in vitro.